The long range objective of this project is to develop methods to modulate opioid peptide action through the peptidases which inactivate them. A major focus has been on the enzyme neprilysin (enkephalinase) which is involved in regulating extracellular enkephalin levels available for receptor binding. Studies will focus on the regulation of expression of multiple neprilysin mRNAs which differ in their 5' untranslated sequence, and are generated from different non-coding exons utilizing different promoters. Expression of neprilysin mRNAs in rat brain will be studied by in situ hybridization and compared to opiate receptor distribution. The effect of different 5' untranslated sequences on mRNA stability and translational efficiency will be determined to see if these factors contribute to the levels of neprilysin mRNA and enzyme activity in different cells. Transcriptional regulation of the neprilysin gene will be studied by comparing the promoter activity of fragments of the gene in cells expressing neprilysin compared to those that do not. Of particular interest will be regulatory elements upstream of the three non-coding exons, since these exons are expressed in a cell specific manner. A transgenic mouse model will be used to determine if cell specific elements identified by cell transfection analysis are functional in vivo Another phase of this project will be to study two newly identified peptidases from the EL-4 thymoma cell line; one cleaves dynorphin B to give Leu-enkephalin-Arg6, and the other cleaves Beta-endorphin to yield gamma-endorphin. These peptidases may be important in modulating opioid peptide actions through intracellular and/or extracellular processing. The cDNAs for these peptidases will be cloned. Specific antisera to each will be generated. These reagents will be used to determine the expression of these enzymes in various tissues and to determine their relationship to previously described enzymes with the same activity. The localization of these enzymes in secretory granules and their co- localization with reaction products will be examined. The mechanism of induction of the dynorphin B cleaving enzyme by morphine will be studied in EL-4 or SHS-Y5Y cells, focusing on whether this is translational or post-translational regulation. Specific inhibitors of the Beta-endorphin metabolizing enzyme will be generated and used to study the role of the enzyme on Beta-endorphin metabolism in brain slices. Attempts will be made to obtain the crystal structure of two peptidases acting on opioid peptides; the puromycin sensitive aminopeptidase and a bacterial neprilysin. High level expression of their cDNAs in E. coli will serve as a source of recombinant enzyme for crystallization trials.